It has been known that certain cationic lipids, e.g., DOTMA (Felgner, Adv. Drug Delivery Rev. 5:163, 1990), can improve the uptake kinetics of nucleic acids in some types of animal cells. Unfortunately, the commercially available preparations of cationic lipids are frequently cytotoxic at concentrations necessary for achieving cellular uptake. In addition, these compounds are not always effective for delivering material to different types of cell populations.
There are a number of commercially available cationic lipids purported to improve the cellular entry of nucleic acids. The prototype compound, DOTMA (Felgner), has been widely used for improving transfection efficiency of cells. This compound is the active ingredient of the commercially available liposomal preparation Lipofectin (Gibco-BRL). Gibco-BRL also markets Lipofectace and Lipofectamine for the same purpose. Boehringer Mannheim supplies DOTAP, and Promega markets Transfectam. Most of these preparations have stringent requirements for administration to cells (e.g., a requirement for a low serum medium, specificity of cell types, quality of nucleic acids) and many are cytotoxic. Gao and Huang (Biochem. Biophys. Res. Comm. 179:280, 1991) have described a cholesterol-containing cationic lipid that is used as a component of liposomes with the potential to enhance the cellular uptake of nucleic acids. However, the structure of the cationic group and the mode of usage of the compound are different from the compounds of the present invention. An amino-sterol, squalamine, isolated from shark tissues, has been shown to be a broad-spectrum antibiotic by Moore et al. (Proc. Natl. Acad. Sci. 90:1354, 1993). This compound is a cationic steroid in which an anionic bile salt intermediate is linked to spermidine. Moore et al. did not suggest any use of the compound in improving nucleic acid delivery into cells. We are not aware of other publications that describe compounds identical or similar to the ones in this disclosure.
There is a real need in the field of nucleic acid therapeutics to develop nonviral uptake enhancement reagents that are nontoxic and effective in a variety of cell types. The numerous commercial preparations are widely perceived to be ineffectual or of limited value for most aspects of nucleic acid delivery. The present invention addresses this problem by the synthesis of simple, relatively nontoxic cationic compounds that improve the cellular and nuclear delivery of nucleic acids.